Turkish Journal of Agriculture - Food Science and Technology, 6(12): 1684-1689, 2018

Turkish Journal of Agriculture - Food Science and Technology Available online, ISSN: 2148-127X www.agrifoodscience.com,

Turkish Science and Technology

Evaluating an Interspecific annuus × Helianthus nuttallii Line for Use in Sunflower Breeding Program

Roumiana Vassilevska-Ivanova1*, Ira Stancheva2, Maria Geneva2, Zoya Tcekova1

1Department of Applied Genetics and Biotechnology, Institute of Physiology and Genetics, Bulgarian Academy of Sciences Akad. Georgy Bontchev Str., Bl 21, 1113 Sofia, Bulgaria 2Department of Plant Mineral Nutrition and Water Relation, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences Akad. Georgy Bontchev Str., Bl 21, 1113 Sofia, Bulgaria A R T I C L E I N F O A B S T R A C T Interspecific cross was made between the common sunflower inbred line HA89 and an Research Article accession of wild Helianthus nuttallii (2n=2x=34) resistant to the most harmful disease complex and tolerant to drought and high temperature. The recombinant inbred line was a Received 13 June 2017 BC1F15 progeny. The most remarkable feature observed was the vigorous plant habit that Accepted 05 June 2018 manifests itself in measurable morphological characters such as increment plant height, stem diameter and diameter of the head. The line was non-branched (monocephalic) Keywords: without anthocyanin pigmentation and possessed good agronomic characteristics. Along Antioxidants with the morphological and reproductive traits, some biochemical characteristics related Enzymes to antioxidant activity were associated to wide hybridization. The overall characteristics Gene Transfer of HA-Hnutt line make it a useful plant material for research on interspecific Pre-breeding Line hybridization in Helianthus genus. Wide Hybridization *Corresponding Author: E-mail: [email protected]

DOI: https://doi.org/10.24925/turjaf.v6i12.1684-1689.1361

Introduction During the last few years, there has been an increasing In the course of our study on wide hybridization in interest in the use of wild sunflower relatives in particular sunflower, we obtained a number of recombinant inbred breeding programs which aim was providing useful lines distinguishing from both parental species. Notably, materials to support practical breeding strategies. To some of these lines could be used as a pre-breeding plant produce potential new cultivars, interspecific and material in many modern breeding programs that make intergeneric hybridizations have been performed between substantial use of wild relatives. common H. annuus and its wild relatives (Faure et al., Herewith, we report the results of interspecific 2002; Breton et al., 2012; Vassilevska-Ivanova et al., hybridization Helianthus annuus × Helianthus nuttallii, in 2013; Vassilevska- Ivanova et al., 2014; Vassilevska- particular, development of a recombinant inbred line Ivanova et al., 2015; Liu et al., 2017; Seiler et al., 2017). produced by conventional cross. The effect on antioxidant Helianthus is a diverse genus comprised of 51 species capacity as well as the content of the antioxidants such as with 14 annual and 37 perennial, all native to North ascorbate, tocopherols, phenols and flavonoids, and America (Shilling, 2006). The very narrow genetic base antioxidant enzymes was investigated. of common sunflower has been broadened by the infusion of genes from wild species, which continue to be a Materials and Methods valuable source of desirable agronomic traits. Helianthus nuttallii T. & G. (2n=2x=34) (Nuttal sunflower) was Plant Material involved in our hybridization program since it appears to The interspecific line H. annuus × H. nuttallii display an immune tolerance to Sclerotinia in USDA originated from pollination of cytoplasmic male sterile genetic stocks (Feng et al., 2007; Seiler et al., 2017), (cms) line HA89 (female), a public oilseed sunflower resistance to the most harmful disease complex (Seiler inbred maintainer line released by US Department of and Marek, 2011), and also it has shown to have drought Agriculture–Agricultural Research Service, with bulked tolerance caused by water deficit under field condition pollen from wild perennial Helianthus nuttallii (male). (Seiler et al., 2017). The population of H. nuttallii was grown from seeds Vassilevska-Ivanova et al., / Turkish Journal of Agriculture - Food Science and Technology, 6(12): 1684-1689, 2018 originally obtained from the experimental garden located measured as the increase in absorbance recorded at 470 at Bloomington (Indiana), USA. The cross was made nm. The enzyme activity was expressed as nmol H2O2 mg using the conventional hybridizing method. Each protein-1 min−1 (Plewa et al. 1991). Ascorbate peroxidase experiment was carried out on head that had been (APX) (EC 1.11.1.1) activity was determined measured protected from foreign pollen by bagging. First- according to the method of Nakano and Asada (1981). generation hybrid were verified using The concentration of oxidized ascorbate was calculated morphological and cytological methods, and F1 hybrids by the decrease in absorbance at 290 nm. Enzyme activity were back-crossed to common sunflower to obtain BC1. was quantified using the molar coefficient for −1 −1 Some BC1 progeny revealed a vigorous phenotype which ascorbate (2.8 mM cm ), and was expressed as moles was considered as valuable for further breeding. These of ascorbate oxidized per milligram of protein per minute plants were selected for and fixed after the BC1 to (McKersie and Leshem 1994). Soluble protein content produce an interspecific line (namely HA-Hnutt). Seeds was determined by the method of Bradford (1976) using from advanced plant generations were produced after self- bovine serum albumin as a standard. pollination under a bag. The plants growth conditions that were employed have already been described (Vassilevska- Antioxidant Capacity Ivanova and Naidenova, 2005). The line represented here The total antioxidant capacity in fresh was is F15 progeny of selfed plants; it was raised in the measured from bleaching of the purple coloured methanol Experimental Field of the Institute of Plant Physiology solution of free stable radical by the method of Tepe et al. and Genetics, Sofia, Bulgaria (located at 42º50´ N, 23º00´ (2006). Antioxidant capacity of the sample was calculated E, 595 m above the sea level) during 2011-2016. Seeds using the equation: were sown in 5 m rows spaced 0.70 cm apart with three replications. The sowings were performed in late-April. % Antioxidant activity (I) = (Ablank-Asampe/Ablank)×100 Conventional management practices were used. Where Ablank is the absorbance of the control sample Phenotypic Observations (containing all reagents except the test compound), and The morphological traits were recorded of thirty Asample is the absorbance of plant extracts. random plants for hybrid line and its parent. Methods The concentration of total phenols in the extracts was used in this investigation for hybridizing plants, fertility measured spectrofotometrically by Folin-Ciocalteu tests and morphological comparisons are the same as method and calculated as caffeic acid equivalents (Pfeffer those described in previous report (Vassilevska-Ivanova et al. 1998). The results were expressed in milligrams of and Naidenova, 2005). The following phenotype traits caffeic acid per gram of dry weight. The total flavonoids were recorded: plant height (cm), head diameter (cm), content was measured spectrofotometrically by the days to flowering (50%), days to maturity (50%), pollen method of Zhishen et al. (1999) using a standard curve staining using the acetocarmine test, thousand seeds with catechin as the standard. Total flavonoids content weight (TSW) (g), and the kernel ratio (%) using the was expressed in milligrams of catechin equivalents per equation: Kernel ratio (%) = kernel weight (g)/grain gram of dry weight. weight (g) × 100. Morphological floral characteristics included the number of ray , the length of the Statistical analysis corolla of ray flowers (cm), and the width of the corolla of Data were subjected to one-way ANOVA analysis of ray flowers (cm). All floral characteristics were measured variance for comparison of means, and significant at the end of anthesis. differences were calculated according to Fisher LSD test at the 5% level using a statistical software package Antioxidant Enzyme Analyses (Statigraphics Plus, version 5.1 for Windows). Enzyme extracts were prepared by homogenizing plants tissue in a pre-chilled mortar in 20 ml chilled Results extraction buffer (pH 7.8). Extracts were then centrifuged at 12 000 g for 30 min at 5°C. Enzyme assays were Phenotype Characteristic and Agronomic Traits conducted immediately following extraction. Superoxide The morphological features of both parental genotypes dismutase (SOD) (EC 1.15.1.1) was measured by and the selected line (H. annuus × H. nuttallii) were photochemical method described by Giannopolitis and assessed (Table 1). In many respects the developing Ries (1977). Assays were carried out under illumination. hybrid line revealed intermediacy to the parental species. One unit of SOD activity was defined as the amount of At maturity, the all plants definitely distinguished in enzyme required to cause 50% inhibition of the rate of p- comparison with its parents showing vegetative vigor and nitro blue tetrazolium chloride reduction at 560 nm. increment in the growth in height and in the head Catalase (CAT) (EC 1.11.1.6) activity was assayed in a diameter (Table 1). The leaves were usually well method following Beers and Sizer (1952) with minor developed, dark green, mostly alternate of H. annuus type modifications. Activity was determined by following with deltoid shape and entire margin. As the whole, -1 decomposition of H2O2 (extinction coefficient, 39.4 mM line HA-Hnutt had more above ground vegetative cm-1) at 240 nm. The enzyme activity was expressed in biomass and smaller seeds than H. annuus. The time to -1 -1 catalytic units-mmol H2O2 mL min per mg protein. bloom (50 %) was accelerated by 10% in the hybrid Guaiacol peroxidase (GPO) (EC 1.11.1.7) activity was plants compared with the cultivated sunflower line. The determined according to Urbanek et al. (1991). The earlier flowering may be attributed to developmental oxidation of guaiacol in the presence of H2O2 was changes that presumably resulted after interspecific 1685

Vassilevska-Ivanova et al., / Turkish Journal of Agriculture - Food Science and Technology, 6(12): 1684-1689, 2018 hybridization, suggesting that it should be possible to annuus HA 89), and from 25.928 to 46.779 µmol Fe2+g-1 breed for the sunflower plants with reduced crop DW in the flowers, respectively. Both DPPH and FRAP production time (Warner and Walworth, 2010). methods together give a good estimate of the antioxidant Furthermore, the line HA-Hnutt has a high degree of potential of the studied plants, acting by different pollen stainability (about 93%) which is a measure in mechanisms. In the current study, the total antioxidant hybridization studies as a proxy for true pollen viability; activity of leaves and flowers extracts measured by FRAP the pollen grains are typically formed and well stained was found to be much lower than either parental samples. (Table1). Alternatively, the level of antioxidants as measured by In common sunflower, H. annuus, the number of outer DPPH was higher in the hybrid line compared with the flowers of the head (ray or ligulate flowers) can be common sunflower line and wild parent H. nuttallii. considered to be a specific qualitative trait characterizing The amount of total phenolics varied slightly in plant putative hybrids (Fambrini et al., 2003). In our case, the materials and ranged from 6.647 to 8.849 mg of caffeic plants produced and flowers with normal acid/g dry sample in the leaves, and from 5.996 to 8.144 structure intermediate in size (smaller than H. annuus but mg of caffeic acid/g dry sample in the ray flowers, larger than H. nuttallii); differences in the number of ray respectively (Table 2). The highest amount was found in flowers and width of corolla of ray flowers became HA-Hnutt line, and the lowest in the cultivated sunflower apparent between both parents (H. annuus and H. H. annuus. The flavonoid contents in the leaves and ray nuttallii) and the selected line, thus indicating that the flowers of both parents and hybrid line are given in Table change in the floral characteristics in hybrid plants was 2. In the leaves, HA-Hnutt line had higher value of 13.843 restricted to ray flowers (Table 1). mg catechin/g dry sample and H. annuus had the lowest value of 11.819 mg catechin/g dry sample, respectively. Antioxidant Capacity and Antioxidant Enzymes The difference in the content of tocopherols is clearly Among the all three genotypes tested, the DPPH expressed in the leaves of HA-Hnutt line and between radical scavenging activity varied from 61.15 to 93.12% parents as H. annuus has markedly low content (Table 2). in the leaves, and from 78.22 to 93.48%, in the flowers In hybrid line, total ascorbic acid (vitamin C) (HA 89 line and hybrid line), respectively (Table 2). concentration in the leaves as well as in the ray flowers FRAP of investigated genotypes varied from 11.060 to was significantly lower than in common sunflower and 41.883 µmol Fe2+g-1 DW in the leaves (line and H. wild H. nuttallii (Table 2).

Table 1 Agronomic characteristics of H. annuus HA 89, H. nuttallii and interspecific line H. annuus × H. nuttallii H. nuttallii Line (hybrid) H. annuus HA 89 Characters mean ± SD** mean ± SD mean ± SD Flowering Days to bloom (50%) 70 64 71 Self-compatibility (%) 50 40 0 Pollen staining (%) 93 93 pollenless Plant height at flowering (cm) 245.0 ± 3.95 185.8 ± 1.15 153.6 ± 0.81 lim 235-255 182-190 145-160 Number of branches highly branched 0 0 lim Stem width, cm 1 ± 0.01 4.6 ± 0.06 3.2 ± 0.02 lim 0.9 - 1.1 4.5 - 4.8 3-3.8 Floral morphology Flower colour sunny yellow sunny yellow sunny yellow Disc colour light light light Number of ray flowers 15.33 ± 0.61 29.8 ± 0.6 47.1 ± 1.23 lim 16-22 27-33 42-54 Length of the corolla of the ray flowers (mm) 1.77 ± 0.03 5.82 ± 0.29 9.54 ± 0.11 lim 1.7-1.9 5.4-6.4 8.9–9.1 Width of the corolla of the ray flower (mm) 0.75 ± 0.02 1.93 ± 0.04 2.49 ± 0.04 lim 0.7 - 0.8 1.0 - 1.2 2.3 - 2.7 Number of leaves 21.4 ± 0.78 31 ± 1.2 lim 19-28 25-37 Maturity *Head diameter (cm) 1.23 ± 0.02 30.9 ± 0.55 24.2 ± 0.36a lim 1.2-1.3 5-7 21-26 Days to maturity (50%) 170 139 162 Post harvest 1000 seed weight (g) 3.02 ± 0.46a 63.6 ± 1.07 77.4 ± 2.38 Kernel ratio (%) 0 40.09 66.92 *-diameter of apical head, **SD=standard deviation

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Table 2 Antioxidant capacity of an interspecific line H. annuus x H. Nuttallii* FRAP (µmol Fe²+ g-1 DW-¹ ) DPPH (%) Parents and hybrid Leaves Ray flowers Leaves Ray flowers H. annuus 41.883a 46.779bc 61.15a 78.22a H. nuttallii 33.996b 41.702b 83.00b 83.09a Hybrid 11.060a 25.928a 93.12bc 93.48ab Phenols (mg/g DW) Flavonoids (mg/g DW) Leaves Ray flowers Leaves Ray flowers H. annuus 6.647a 5.996a 11.819a 8.309a H. nuttallii 7.200ab 7.981b 12.505b 13.534a Hybrid 8.849b 8.144b 13.843c 9.601a WS-AOC (µ mol g-¹ DW -¹ ) LS-AOC (µ mol g-¹ DW -¹ ) Leaves Ray flowers Leaves Ray flowers H. annuus 248.471b 495.059b 0.650a 0.800a H. nuttallii 473.412c 611.765c 1.150b 0.950a Hybrid 183.529a 103.059a 1.300c 0.950a *Data were reported as means ± standard error. Standard errors were represented as vertical bars which were the mean of three values (n=3); letters in common indicate no significant differences assessed by Fisher LSD test (P≤0.05) after performing ANOVA.

Table 3. Antioxidant enzymes of an interspecific line H. annuus x H. Nuttallii* CAT (units mg pr-1 min-1) GPO (units mg pr-1 min-1) Parents and hybrid Leaves Ray flowers Leaves Ray flowers H. annuus 16.250b 13.140b 92.498a 31.010a H. nuttallii 5.691a 3.513c 476.095c 200.526b Hybrid 15.286a 4.651a 294.057b 23.217a APX (µmol ASC mg pr-1 min -1 SOD (units mg pr-1 min-1) Leaves Ray flowers Leaves Ray flowers H. annuus 0.720b 0.400c 56.422b 36.154b H. nuttallii 0.109a 0.158b 13.253a 57.385c Hybrid 0.068a 0.080a 13.527a 9.803a *Data were reported as means ± standard error. Standard errors were represented as vertical bars which were the mean of three values (n=3); letters in common indicate no significant differences assessed by Fisher LSD test (P≤0.05) after performing ANOVA.

Determination of the Level of CAT, SOD, GPO, and improvements (Davey et al, 2010; Breton et al, 2012; APX in the Three Sunflower Genotypes Zhang et al., 2017). Wide hybridization has been Analysis of CAT activity revealed differences performed between common sunflower, H. annuus and its between three genotypes, which were evident both in the wild relatives to produce enhanced sunflower germplasms leaves and ray flowers (Table 3). The most striking of with high yield, high oil content, and desirable fatty acid these differences was the activity of CAT in perennial H. concentration, as well as novel resistance genes for nuttallii. GPO activities in the leaves for three selected diseases and insects (Seiler and Marek, 2011; Kaya, 2014; genotypes were higher than that these in the ray flowers, Zhang et al., 2017). In this view, hybridization may play a despite of the almost equal phenolic content in both plant creative role in providing the genetic “row material” organs (Table 3). The highest value was established in the required to support practical breeding strategies, and also leaves of H. nuttallii. However, the GPO activity of to study the genetic relationships between species in the hybrid line was intermediate of two parental genotypes. In genus Helianthus. Herewith, we examined the late- common sunflower H. annuus, SOD-activity was generation fitness consequences of hybridization between statistically higher than activities recorded in wild the common sunflower, H. annuus and wild perennial sunflower and HA-Hnutt line (Table 3). There was no diploid H. nuttallii. According to Johansen-Morris and substantial difference in the level of SOD in the leaves Latta (2006), the fitness of later generation recombinant sample of H. nuttallii and hybrid line. However, in ray- inbred lines (RILs) derived from the cross reflects both flowers of hybrid plants, the level of SOD was strongly the loss of early generation heterozygosity as well as reduced compared with both parental genotypes. disruption of any collapsed gene complexes present in the Common sunflower showed the highest APX activity in parents. Herewith, we have presented a recombinant the leaves when compared with H. nuttallii and hybrid inbred line (RIL) easily distinguished from both common line (Table 3). Similar trend revealed the APX activity in sunflower and wild perennial parents that will likely be the ray flowers. Hybrid, however, revealed the lowest used in sunflower breeding. APX activity in the leaves than either parent. As in other studies (Faure et al., 2002; Vassilevska- Ivanova et al., 2003; Breton et al., 2012), the interspecific Discussion hybridization affects a wide range of vegetative and reproductive traits. In terms of phenotype, the common In Helianthus crop breeding programs, wild relatives sunflower, H. annuus appeared to be dominant since have been evaluated as genetic resources to develop new hybrid H. annuus × H. nuttallii matched closely the cultivars with potential traits that could lead to female parent for some H. annuus-line morphological 1687

Vassilevska-Ivanova et al., / Turkish Journal of Agriculture - Food Science and Technology, 6(12): 1684-1689, 2018 traits. The most remarkable feature observed was the generalizations of methods for testing of experimental vigorous plant habit that manifests itself in measurable hybrids in the selection studies. morphological characters such as increment plant height, In conclusion, it is possible to produce fertile hybrid stem diameter and diameter of the head, thus, suggesting plants from crosses between common sunflower, H. the pleiotropic effect of hybridization. Further, the growth annuus and wild perennial diploid species H. nuttallii. habit and flower features had a clearly intermediate Morphological and biochemical characters provide character, thus, indicating that characters from both substantial evidence that H. nuttallii is involved in parents are being expressed. Morphological data are hybridization process. Outcomes of hybridization between consistent with several studies in that the morphological both Helianthus species show the formation of a stable intermediacy of the hybrids between the common single headed line with extreme plant habit. Our results sunflower, H. annuus and several of its congeners, suggest that the genetic diversity between two species is including H. mollis (Faure et al., 2002; Vassilevska- not a strong barrier to the hybrid formation. Nevertheless, Ivanova et al., 2005; Breton et al., 2012), H. argophyllus more research is needed to elucidate the real potential of (Vassilevska-Ivanova et al., 1998), and wild H. annuus, wild sunflower germplasms and their introduction into H. petiolaris, H. bolanderi (Vassilevska–Ivanova et al., breeding programs to develop high performance cultivars. 2001) has been documented. Along with the morphological and reproductive traits, Conflict of Interest some biochemical characteristics related to antioxidant activity were associated to wide hybridization. The The authors declare no conflict of interest. comparison of hybrid line and both parental species revealed that antioxidant capacity and the level of References antioxidant enzymes such as CAT, SOD, GPO, and APX were altered by the interspecific hybridization to a Beers Jr RF, Sizer IW. 1952. A spectrophotometric method for different extent. It was observed that, among the analyzed measuring the breakdown of hydrogen peroxide by catalase. biochemical parameters FRAP, ascorbic acid, the amount Journal of Biological Chemistry, 195 (1):133–140. of total tocopherols, CAT, and GPO content were mostly Bradford MM. 1976. A rapid and sensitive method for the intermediate. Among these parameters, the total content estimation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, of tocopherols seems to be of practical interest 72: 248-254. considering its high antioxidant potential. It was Breton C, Gil A, Wargnier J, Seriyes H, Berville A. 2012. established that the sunflower oil is the richest in α- Transfer of architectural traits from perennial Helianthus tocopherol which is the main compound among the mollis Lam. to sunflower (H. annuus L.) and localisation of tocopherol homologs with the highest biological activities introgression. Euphytica, 186 (2): 557-572. (Seiler and Marek, 2017). This fact could contribute to Davey MR, Masood J. 2010. Sunflower (Helianthus annuus L.): reinforcing the idea that interspecific hybridization can be genetic improvement using conventional and in vitro a potential tool to improve vitamin E status of the technologies. Journal of Crop Improvement, 24 (4): 349– sunflower oils rich in monounsaturated fatty acids. 391. Faure N, Serieys H, Cazaux E, Kaan F, Berville A. 2002. Partial However, further studies are needed to characterize the hybridization in wide crosses between cultivated sunflower composition of hybrid oil and to assess its potential as a and the perennial helianthus species H. mollis and H. source of health-promoting bioactive compounds. orgyalis. Annals of Botany, 89 (1): 31-39. The present study indicated significant differences in Fambrini M, Bertini D, Pugliesi C. 2003. The genetic basis of a antioxidant enzymes activity among three investigated mutation that alters the floral symmetry in sunflower. genotypes. It was reported that very few studies have Annals of Applied Biology, 143 (3): 341-347. attempted to determine the mechanisms behind the Feng Z, Liu X, Cai G, Seiler J, Gulya TJ, Rashid KY, Jan CC patterns of chemical variation, thus reducing ability to (2007). Transferring Sclerotinia resistance genes from wild predict which hybrids will be more or less variable Helianthus into cultivated Sunflower. J North Dakota State University, USDA-ARS, ND 58105: 1-3. (Orians, 2000). Also, genetic studies to ascertain whether Giannopolitis CN, Ries SK. 1977. Superoxide dismutases I. the pattern of chemical variation arose as a direct by- Occurrence in higher plants. Plant Physiology, 59(2): 309- product of hybridization or whether they evolved via 314. other mechanisms, for example, the type of divergence is Johansen-Morris AD, Latta RG. 2006. Fitness consequences of necessary to be performed. In the leaf samples of HA- hybridization between ecotypes of Avena barbata: hybrid Hnutt line, the total phenolic and flavonoids content breakdown, hybrid vigor, and transgressive segregation. altered to a different extent (Table 2); the total phenolic Evolution, 60 (8): 1585-1595. content and the flavonoids were higher than these in both Kaya Y. 2014. Sunflower. In Alien Gene Transfer in Crop parents. In the current study, the antioxidant level of Plants, Pratap, A. and J. Kumar, (Eds.). Springer, v. 2: 281– 300. phenols and flavonoids is an indication that both Liu Z, Seiler GJ, Gulya TJ, Feng J, Rashid KY, Cai X, Jan CC. characteristics might be hardly useful as biochemical 2017. Triploid production from interspecific crosses of two diagnostic markers for preliminary evaluation of the diploid perennial Helianthus with diploid cultivated hybridization events. So, in relation to the sunflowers (H. sunflower (Helianthus annuus L.). G3 (Bethesda), 37 (4): annuus and its wild relatives), the identification of 1097-1108. specific biochemical markers that verified the hybrid McKersie BD, Leshem YY. 1994. Stress and stress coping in origin of the plants is needed to be investigated. Thus, we cultivated plants. Kluwer Academic Publishes, Dordrecht. can come closer to mechanisms of wide hybridization and pp. 256

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